MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY

Abstract

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY Ryuta Watanabe*, Masashi Maekawa, Miki Hieda, Tomohiko Taguchi, Noriyoshi Miura, Tadahiko Kikugawa, Shigeki Higashiyama, and Takashi Saika Ryuta Watanabe*Ryuta Watanabe* More articles by this author , Masashi MaekawaMasashi Maekawa More articles by this author , Miki HiedaMiki Hieda More articles by this author , Tomohiko TaguchiTomohiko Taguchi More articles by this author , Noriyoshi MiuraNoriyoshi Miura More articles by this author , Tadahiko KikugawaTadahiko Kikugawa More articles by this author , Shigeki HigashiyamaShigeki Higashiyama More articles by this author , and Takashi SaikaTakashi Saika More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: SPOP, speckle-type POZ protein, is a substrate recognizing receptor of the Cullin-3/RING ubiquitin E3 complex. Heterozygous point mutations in the substrate-binding domain of SPOP have been found in 10-15% of human prostate cancer patients. Although SPOP is essential for a DNA repair in exogenous DNA damage stresses, it still remains unclear whether SPOP is involved in the process of a DNA repair in endogenous DNA damage stresses. In this study, we focus on relationships the functions of SPOP, especially those involved in topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2), to the DNA-protein crosslink repair from endogenous DNA damage in prostate cancer cell lines. METHODS: We first examined the level of γH2AX, a marker of DNA breaks in various prostate cancer cell lines; C4-2, LNCaP, PC3, DU145 upon SPOP knockdown (KD). To elucidate the site of action of SPOP during DNA repair, we treated control or SPOP-KD C4-2 cells with topoisomerase (TOP) inhibitors, which inhibit DNA repair. To investigate functions of SPOP in the regulation of TOP, we analyzed protein expression and cellular localization of TOPs by western blotting and immunofluorescence staining. We also measured the amounts of TOPs which covalently bound on DNA by cesium chloride-density gradient centrifugation. We finally detected the level of enzymes (TDP1, TDP2 and MRE11) which liberate TOPs from the TOP-DNA cleavage complex. RESULTS: We found that SPOP KD in androgen receptor (AR)-positive prostate cancer cells increased the amount of γH2AX. Because the level of γH2AX in SPOP-knockdown cells was not additive by treatment of TOP inhibitors, it was suggested that SPOP could act upstream of TOP1 and TOP2-dependent pathways. Immunofluorescence staining indicated that TOP2A, but not TOP1, was accumulated in the nuclei by SPOP knockdown. By cesium chloride-density gradient centrifugation, we found that SPOP KD increased the amount of TOP2A on genomic DNA. The amount of TDP1 and TDP2, which liberate TOP2A from the TOP2A-DNA complex was decreased by SPOP knockdown. We also confirmed that the increase of γH2AX in SPOP-KD C4-2 cells was dependent on both AR and TOP2A. CONCLUSIONS: Our results suggest that SPOP is involved in the DNA-protein crosslink repair process through the elimination of TOP2A from the TOP2A cleavage complex in prostate cancer cell lines. These results imply that TOP inhibitor might be effective to AR-positive, SPOP-wild type prostate cancer. Also, PARP inhibitor might be effective to SPOP-mutated prostate cancer. Source of Funding: JSPS KAKENHI Grant Number 19K18613 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e767-e767 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuta Watanabe* More articles by this author Masashi Maekawa More articles by this author Miki Hieda More articles by this author Tomohiko Taguchi More articles by this author Noriyoshi Miura More articles by this author Tadahiko Kikugawa More articles by this author Shigeki Higashiyama More articles by this author Takashi Saika More articles by this author Expand All Advertisement PDF downloadLoading ...